Farnesylated peptides in model membranes: a biophysical investigation

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.     

Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling.     

Mechanisms of DNA bending

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.     

Structural Analysis of HMGD-DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.     

Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.     

高迁移率族蛋白与真核基因表达调控

高迁移率族蛋白 (high mobility group protein , HMG) 是一系列的染色质相关蛋白,广泛存在于真核生物细胞中,含量丰富,因其在聚丙烯酰胺凝胶电泳中的高迁移率而得名 . HMG 蛋白家族可分为 HMGB 、 HMGA 和 HMGN 三类亚家族,各亚家族有其特征的结构域,这些结构域介导了 HMG 和 DNA 或染色质相关区域的相互作用 . 现已发现这些蛋白质具有多种重要生物学功能,其中几乎所有 HMG 都可以通过修饰、弯曲或改变染色质 /DNA 的结构,促进各种蛋白质因子形成大分子复合物来调节基因转录 .     

Selective DNA bending by a variety of bZIP proteins.

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.     

Asymmetric DNA bending induced by the yeast multifunctional factor TUF

TUF is a yeast regulatory factor that binds to conserved DNA sequence elements involved in gene activation or silencing as well as in telomere function. Using gel electrophoresis analyses, we show here that TUF induces DNA bending at a site located upstream of the recognition sequence (rpg box). Several point mutations in the rpg box reduced TUF binding strength without affecting the extent of bending. Selective proteolysis of TUF.DNA complexes further suggested the existence of two separate protein domains involved in DNA bending and specific DNA recognition. DNA bending may be an important feature of multifunctional factors that could help them to recruit other proteins for the formation of multiprotein complexes.     

Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA

Background:  

The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences.